Micelle and Nanotape Formation of Benzene Tricarboxamide Analogues with Selective Cancer Cell Cytotoxicity

Analogues of benzene-1,3,5-tricarboxamide bearing combinations of different alkyl chains (dodecyl to octadecyl) and ester-linked PEG (polyethylene glycol) chains are shown to self-assemble into either micelles or nanotapes in aqueous solution, depending on the architecture (number of alkyl vs PEG chains). The cytotoxicity to cells is selectively greater for breast cancer cells than fibroblast controls in a dose-dependent manner. The compounds show strong stability, retaining their self-assembled structures at low pH (relevant to acidic tumor conditions) and in buffer and cell culture media.


Materials
Reagents and solvents were purchased from Sigma Aldrich and used without further purification. Tetrahydrofuran (THF) was distilled under a nitrogen atmosphere from sodium benzophenone. Chloroform was distilled under a nitrogen atmosphere from calcium chloride.
Synthesis details and characterization data are provided below.

Methods
Thin layer chromatography (TLC) was performed on aluminium sheets coated with Merck silica gel 60 F24. Spots were visualised under ultra-violet light (254 nm) with potassium permanganate as the visualising agent. Column chromatography was performed using Merck silica gel 60 (40-63 μm particle size) and a mobile phase as specified. Melting points were recorded using a Stuart SMP10 melting point apparatus. 1 H NMR and 13 C NMR spectra were recorded using either CDCl3 or DMSO-d6 as solvent on either a Bruker Nanobay 400 or Bruker DPX 400 operating at 400 MHz for 1 H NMR or at 100 MHz for 13 C NMR. Mass spectrometry analysis was carried out using a Thermo-Fisher Scientific Orbitrap XL LC-MS. Fourier-Transform Infrared (FT-IR) spectra were measured using a Perkin-Elmer Spectrum 100 FTIR spectrometer set up with an ATR attachment allowing rapid recording of FTIR spectra that were scanned 32 times over the range 900−4000 cm −1 .

Cryogenic-Transmission Electron Microscopy (Cryo-TEM).
Imaging was carried out using a field emission cryo-electron microscope (JEOL JEM-3200FSC), operating at 200 kV. Images were taken in bright field mode and using zero loss energy filtering (omega type) with a slit width of 20 eV. Micrographs were recorded using a Gatan Ultrascan 4000 CCD camera. The specimen temperature was maintained at -187 °C during the imaging. Vitrified specimens were prepared using an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon copper grids with a hole size of 3.5 m. Just prior to use, grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner and then transferred into the environmental chamber of a FEI Vitrobot at room temperature and 100 % humidity. Thereafter 3 l of sample solution was applied on the grid and it was blotted twice for 5 seconds and then vitrified in a 1/1 mixture of liquid ethane and propane at temperature of -180 °C. The grids with vitrified sample solution were maintained at liquid nitrogen temperature and then cryo-transferred to the microscope. 1 μΜ, 10 μΜ and 100 μΜ. After 67 h of incubation, a 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) solution (5 mg/mL in phosphate-buffered saline (PBS)) was added (20 μL per well) and allowed to incubate for 5 h (total of 72 h of incubation). After this, the solution was removed from each well and replaced with 100 μL of dimethyl sulfoxide (DMSO4) per well to dissolve the formazan crystals. Plates were incubated for 30 min and then analysed using a UV microplate reader (λ = 560 nm). Results are reported as a percentage cell viability compared with control (untreated values). All assays were performed in triplicate.

Synthesis of 5-methoxycarbonyl-benzene-1,3-dicarboxylic acid (3).
Trimethyl benzene-1,3,5-tricarboxylate 2 (5 g, 19 mmol) and 2.3 eq. NaOH (1.7 g, 44 mmol) were dissolved in MeOH (350 mL). The mixture was stirred for 24 hours under reflux at 85 °C, after which the mixture was allowed to cool down to room temperature. Thereafter, the mixture was concentrated to about 150 mL under vacuum, poured in 400 mL 1 M HCl in a separation funnel of 1 L and extracted with diethyl ether (3 x 300 mL). The organic layers were collected and the solvent was removed under vacuum. The crude product was recrystallized twice from ethyl acetate and obtained as a white solid (2.9 g, 61%); IR (ATR) ν/cm Synthesis of methyl-3,5-bis-chlorocarbonyl-benzoate (4). 5-methoxycarbonylbenzene-1,3dicarboxylic acid (1.0g, 4.46 mmol) was dissolved in 30 mL of dry THF under a nitrogen atmosphere and a catalytic amount of DMF (~ 2 droplets). To this solution was added dropwise a solution of 2.5 eq. oxalyl chloride (0.95 mL, 11.15 mmol) in 10 mL of dry THF. The reaction was stirred for 90 min at room temperature (completion checked with 1 H-NMR), after which the THF was removed in vacuo and the excess of oxalyl chloride was removed by coevaporation with toluene (3 x 50 mL). The product was obtained as a yellow oil (1.12g, 97%).

Synthesis of methyl-3,5-bis-n-Alkylaminocarbonyl-benzoate (5).
A solution of 2 eq. Alkylamine and 3 eq. triethylamine was dissolved in dry CHCl3 in ice bath under a nitrogen atmosphere. To this solution was added dropwise a solution of methyl-3,5-bis-chlorocarbonylbenzoate 4 (1 eq.) in dry CHCl3. The reaction was stirred for 24 hours at room temperature under nitrogen atmosphere. Then, the solvent was removed under vacuum and the resulting crude product was purified with a short silica-gel (CH2Cl2: MeOH; 100:2) obtained a white product. Synthesis of 3,5-bis-n-Alkylcarbamoyl benzoic acid (6). 1 eq. methyl-3,5-bis-n-Alkylaminocarbonylbenzoate and 2 eq. of NaOH (4M) were dissolved in methanol. The solution was stirred and reflux at 85 ˚C for 24h after which the solution was cooled and poured into 300 mL H2O that acidified with 1 M HCl. A white powder precipitated and was isolated by filtration. The product was dried under vacuum. General Synthesis for of DAmMPEG550 (7). As a first step 3,5-bis-n-alkylcarbamoylbenzoyl chloride was synthesised following the same procedure as described for 4, 1 eq. of 3,5bis-n-Alkylcarbamoyl benzoic acid 6 and 1.5 eq. of oxalylchloride under nitrogen atmosphere yielding yellowish product. The resulted product was dissolved in dry THF and added slowly S-7 to a solution of Poly (ethylene glycol) methyl ether (Mn 550) 1 eq. and triethylamine 2 eq. in dry THF in ice bath under a nitrogen atmosphere. The reaction was stirred overnight at room temperature under nitrogen atmosphere. Then, the solvent was removed under vacuum and the resulting crude product was purified by column chromatography.
DA10MPEG550. The synthesis of DA10MPEG550 was performed following the same procedure as described for 7, was used Poly (ethylene glycol) methyl ether (Mn 550) (0.35 mL, 0.59 mmol) and triethylamine (0.15 mL, 1.48 mmol) in 15 mL dry THF in 0 ˚C under a nitrogen atmosphere. To this solution, a solution of 3,5-bis-n-decylaminocarbonyl-benzoyl chloride (0.25 g, 0.49 mmol) in 5 mL of dry THF. The reaction was stirred overnight at room temperature under nitrogen atmosphere. Then, the solvent was removed under vacuum and the resulted crude product was purified with silica-gel (CHCl3: MeOH; 100:7) obtain a yellowish oil (0.

Synthesis of 1,3-dimethoxycarbonyl-benzene-5-carboxylic acid (8).
Benzene-1,3,5tricarboxylic acid trimethyl ester 2 (5.00 g, 19.82 mmol) and 4M NaOH (0.95 g, 23.78 mmol) were dissolved in methanol (150 mL) and refluxed for overnight at 85 ˚C. after which the solvent was removed under vacuum and dissolved in CH2Cl2 (300 mL) and extracted with saturated sodium bicarbonate solution (600 mL). The collected aqueous phase was washed with CH2Cl2 and acidified with 1M HCl resulting white precipitate that filtered and dried under vacuum giving white solid Synthesis of dimethyl 5-(chlorocarbonyl) isophthalate (9). 1,3-dimethoxycarbonylbenzene-5-carboxylic acid 8 (0.5 g, 2.10 mmol) was dissolved in 25 mL dry chloroform. After addition of a catalytic amount DMF, oxalyl chloride (190 μL, 2.25 mmol) in 10 mL dry chloroform was added drop wise. After stirring for 90 minutes, the excess oxalyl chloride and chloroform were removed in vacuo. The remaining solid was co-evaporated twice with 50 mL toluene to ensure the removal of oxalyl chloride. The product was obtained as a yellow oil

Synthesis of dimethyl 5-(alkylcarbamoyl)isophthalate (10).
A solution of 1 eq. alkylamine and 1.3 eq. TEA was dissolved in dry CH2Cl2 under nitrogen atmosphere at 0˚C. To this solution, a solution of dimethyl 5-(chlorocarbonyl) isophthalate 9 (1 eq.) in dry CH2Cl2 was added drop wise to that and stirred for 24h at room temperature. Then, the solvent was removed under vacuum and the crude product was purified by column chromatography (CH2Cl2: MeOH, 100:1) yielding a white powder.

Synthesis of 5-(alkylcarbamoyl) isophthalic (11).
Dimethyl 5-(alkylcarbamoyl)isophthalate 10 (1 eq.) was dissolved in methanol with 4M NaOH that was stirred and reflux at 85 ˚C for 24 hours. the mixture was cooled to room temperature before being poured to 1M HCl. A white powder precipitate was formed and filtered. The precipitate was recrystallized from ethyl acetate (2 times), leading to a white product.  5, 165.4,135.6, 133.1, 132, 131.1, 40.4, 31.9, 29.7 General Synthesis for of MAmDPEG550 (12). As a first step 5-(alkylcarbamoyl)isophthaloyl dichloride was synthesised following the same procedure as described for 9, 1 eq. of 5-(alkylcarbamoyl) isophthalic 11 and 2.5 eq. of oxalylchloride yielding a yellowish product. The resulted product was dissolved in dry THF and added slowly to a solution of 2 eq. Poly (ethylene glycol) methyl ether (Mn 550) and triethylamine 3 eq. in dry THF in ice bath under a nitrogen atmosphere. The reaction was stirred overnight at room temperature under nitrogen atmosphere. Then, the solvent was removed under vacuum and the resulting crude product was purified by column chromatography (CHCl3: MeOH).       Table S2). Data has been scaled to enable visualization, and only every 5th measured data point is shown.  1   MA10N  MA12N  MA16N  MA18N  DA10N  DA12N  DA16N  DA18N  MA10A  MA12A  MA16A  MA18A  DA10A  DA12A  DA16A  DA18A  MA10B  MA12B  MA16B  MA18B  DA10B  DA12B  DA16B  DA18B   neutral  Table S1. Parameters extracted from the fitting of the SAXS data a at neutral pH. All samples were 1 wt% aqueous solutions.